Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations of these references can be found throughout the specification. Each of these citations is incorporated herein by reference as though set forth in full.
Platelets are critical for hemostasis and are anucleate cellular fragments derived from megakaryocytes (Kaushansky, K. (2008) Blood 111:981-6). Platelets also have important roles in angiogenesis and inflammation (Browder et al. (2000) J. Biol. Chem., 275:1521-4; May et al. (2008) Arterioscler. Thromb. Vasc. Biol., s5-10). Transfusions of platelets are used in diverse clinical settings, including individuals with decreased platelet production, for example, secondary to chemotherapy-induced thrombocytopenia (CIT) (Benjamin et al. (2002) Crit. Rev. Oncol. Hematol., 42:163-71), and those with increased platelet destruction, for example, in patients with disseminated intravascular coagulopathies (Dempfle, C. E. (2004) Thromb. Haemost., 91:213-24). In 2001 in the USA, platelet transfusions totaled 10,196,000 units, an increase of 12.6% from 1999 (Sullivan et al. (2007) Transfusion 47:385-394). The use of single-donor apheresis platelets increased at the same time by 26% to 7,582,000 platelet concentrate units.
While the number of platelet donors is increasing, there is still a significant donor shortage due to the growing population of patients with serious illnesses associated with thrombocytopenia and hemorrhage (Sullivan et al. (2007) Transfusion, 47:385-394). At present, platelet transfusions are limited by their short storage half-life, apheresis requiring a donor to undergo a 2-hour procedure, and units varying significantly both qualitatively and quantitatively (Kaufman, R. M. (2006) Hematology Am. Soc. Hematol. Educ. Progr., 492-6; Heddle et al. (2008) Transfusion 48:1447-589; Garner et al. (2008) Transfusion 48:673-80). Because platelets have to be stored at room temperature, these units have the highest incidence of bacterial contamination (Eder et al. (2007) Transfusion 47:1134-42). Even with leuko-reduction to remove contaminating white cells, there is still a high incidence of inhibitors developing in multiply transfused patients (Seftel et al. (2004) Blood 103:333-9). Thus, the use of donor-derived platelets raises the following concerns: variability of quality and quantity, risk of infectious transmission, short lifespan of stored platelets, bacterial contamination during storage, and development of alloantibodies in multi-transfused patients. These problems highlight a need for new strategies to generate platelets for infusion therapy.
Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes remains incompletely understood. In vitro studies suggest that platelets form nodes at tips of proplatelet strands (Italiano et al. (1999) J. Cell. Biol., 147:1299-1312). However, direct visualization of live calvaria marrow using multiphoton intravital microscopy suggests that megakaryocytes release large cytoplasmic fragments into the vasculature (Junt et al. (2007) Science 317:1767-1770), which must then undergo reorganization into platelets. Studies based on morphologic analysis and quantification of megakaryocyte-like polyploid nuclei in the pulmonary venous system suggested that megakaryocytes release platelets in the lungs (Zucker-Franklin et al. (2000) Am. J. Pathol., 157:69-74). Derivation of platelets from megakaryocytes in culture has been reported (Choi et al. (1995) Blood 85:402-413) but has been difficult to quantitatively upscale. Present day efforts to develop platelets ex vivo have met with very limited and not clinically relevant results. To date, the best published result from infused in vitro produced platelets used irradiated mice with low platelet counts (˜104/μl) (Nishikii et al. (2008) J. Exp. Med., 205:1917-1927). Peak percent donor platelet counts were still only 1%-2%.